Il-4-derived peptides for modulation of the chronic inflammatory response and treatment of autoimmune diseases

ABSTRACT

The present invention relates to small peptides derived from a cytokine, interleukin-4 (IL-4), capable of binding to the IL-4 receptors and inhibiting macrophage activation, and thereby preventing the onset of inflammatory response. The invention further relates to use of said peptides for the production of a medicament for the treatment of different pathological conditions, wherein IL-4 plays a prominent role.

FIELD OF INVENTION

The present invention relates to small peptides derived from a cytokine,interleukin-4 (IL-4), capable of binding to the IL-4 receptors andinhibiting macrophage activation, and thereby preventing the onset ofinflammatory response. The invention further relates to use of saidpeptides for the production of a medicament for the treatment ofdifferent pathological conditions, wherein IL-4 plays a prominent role.

BACKGROUND OF INVENTION

Abnormalities associated with inflammation comprise a large, unrelatedgroup of disorders which underlie a variety of human diseases. Examplesof disorders associated with inflammation include asthma, chronicinflammation, and autoimmune diseases including rheumatoid arthritis.Chronic inflammation is a pathological condition characterised byconcurrent active inflammation, tissue destruction, and attempts atrepair. Rheumatoid arthritis (RA) is a chronic, systemic autoimmunedisorder that causes the immune system to attack the joints, where itcauses inflammation (arthritis) and destruction. It can also damage someorgans, such as the lungs and skin. It can be a disabling and painfulcondition, which can lead to substantial loss of functioning andmobility. It is diagnosed with blood tests (especially a test calledrheumatoid factor) and X-rays.

The inflammatory reaction observed in autoimmune disease involves bothcellular and soluble players. The cause of RA is not known. It involvescomplex interactions of various cells, cytokines and enzymes. Thedisease begins when an inciting antigen gains access to the joint,triggering an immune response. The antigenic stimulus activates CD4+lymphocytes (T-cells). Once CD4+ T-cells become activated, a complexcascade of biological events take place including stimulation ofmacrophages, B-cells, fibroblasts, chondrocytes and osteoclasts.Activated macrophages secrete cytokines, such as interleukin-1 (IL-1),IL-6, IL-8, IL-15 and tumor necrosis factor-α (TNF-α) (Martinez et al.,2008).

Interleukin-4 (IL-4) is secreted by CD4+ T-cells (Th2 cells). It is apleiotropic cytokine, acting on various cell types and tissues. Itsaction on immune cells results in activation and growth of B cells, IgGand IgE production, MHC class II induction, growth and survival of Tcells, Th2 differentiation, enhancement of mast cell growth, enhancementof IL-2 and IL-12-induced interferon-γ (INF-γ) secretion in NK cells,downregulation of C5a and C3a in monocytes and Mo-derived dendriticcells and inhibition of macrophage activation (Agnello et al., 2003;Szehedi et al., 2003; Roland, 2003).

The structure of recombinant human IL-4 has been determined by both NMRand X-ray diffraction methods in several laboratories. It has aclassical 4 helix bundle cytokine structure (Muller et al., 1995). IL-4,like other cytokines, exerts its biological activity by binding to thereceptors on the cell surface. One receptor complex is composed of twocomponents, the IL-4R α chain (IL-4Rα) and the IL-2R γ chain (γc, sharedby the cytokines IL-2, IL-7, IL-9, IL-15 and IL-21), denoted type IIL-4R, whereas the other receptor complex is composed of IL-4Rα and theIL13 α chain (IL-13Rα1), called type II IL-4R. As γ c is expressed onmost hematopoietic and immune cells, IL-4 is assumed to act on thesecells through type I IL-4R. In contrast, expression of IL-13Rα1 islimited to some lineages such as B cells in hematopoietic and immunecells, but ubiquitously detected on non-immune cells (Izuhara et al.,2002). Thus IL4 acts on non-immune cells through type II IL-4R/IL-13R.

Binding IL-4 to its receptor a chain (IL-4Ra) is a crucial event for thegeneration of a Th2-dominated early immune response. The crystalstructure of the intermediate complex between human IL-4 and IL4-BP wasdetermined at 2.3 Å Resolution (PDB ID: 1IAR). It reveals a novelspatial orientation of the two proteins, a small but unexpectedconformational change in the receptor-bound IL-4, and an interface withthree separate clusters of trans-interacting residues (Hage et al.,1999). Crystal structure of the II4-II4r-common gamma ternary complexhas recently been solved (PDB ID: 3BPL; LaPorte et al., 2008).

Recombinant IL-4 has been through several clinical trials. IL-4 has beenshown to be beneficial in patients with psoriasis, effectivelycorrecting imbalances in immune functions (Martin 2003). The safety andtolerability of Escherichia coli-derived recombinant human interleukin-4(rhulL-4) have been evaluated in phase I and phase II studies in humanpatients with a variety of malignancies. Clinical trials havedemonstrated that subcutaneous administration of rhulL-4 is safe andwell tolerated at doses as high as 5 μg/kg/day and as high as 10 μg/kgwhen administered 3 times/week. Although preclinical safety studies incynomolgus monkeys demonstrated a number of adverse effects followingrepeated daily dosing with rhulL-4, similar effects have generally notbeen observed in human patients (Leach et al., 1997). The most commontoxicities were elevated liver function tests, nausea/vomiting/diarrhea,malaise/fatigue, edema, headache, myalgias/arthralgias, andfever/chills. Despite promising preclinical growth inhibitory andimmunomodulatory effects, IL-4 in this dose and schedule showed only lowantitumor activity (Whitehead et al., 1998).

Many human autoimmune and inflammatory diseases are still treated by acombination of corticosteroids and general immunosuppression. A betterunderstanding of the pathogenesis of these diseases has led to therapiesthat are more specific. Among these, the recombinant humanized proteinsare considered as the future therapies. However, drugs based onrecombinant proteins have several disadvantages including highproduction cost, big batch-to-batch variation and denaturation duringstorage.

SUMMARY OF INVENTION

The present invention concerns fragments of IL-4 that can be chemicallysynthesized and used as functional mimetics of IL-4.

The present invention relates to a compound comprising an isolatedpeptide consisting of at most 35 contiguous amino acid residues derivedfrom IL-4 or a variant being at least 70% identical. A compoundcomprising such amino acid sequence is according to the inventioncapable of i) binding to the IL-4 receptor; ii) inhibiting aninflammatory response; iii) inhibiting macrophage activation; iii)activating B-cells; iv) activating growth and survival of T-cells; v)downregulating C5a and C3a in monocytes and dendritic cells, vi)modulating activity of the IL-4 receptor.

Accordingly, another aspect of the invention relates to use of compoundsof the invention as medicaments and for the preparation of medicamentsfor treatment of a condition or disease wherein i) binding to the IL-4receptor; ii) inhibiting an inflammatory response; iii) inhibitingmacrophage activation; iii) activating B-cells; iv) activating growthand survival of T-cells; v) downregulating C5a and C3a in monocytes anddendritic cells, vi) modulating activity of the IL-4 receptor is part ofsaid treatment.

Still, in another aspect a peptide of the invention or a compoundcomprising the peptide may be used for the production of an antibody.Such antibodies will bind an epitope within a peptide of the invention.

The invention further relates to pharmaceutical compositions comprisinga peptide of the invention, or an antibody capable of recognising anepitope within a peptide of the invention.

The invention also concerns a method of treatment of conditions whereini) binding to the IL-4 receptor; ii) inhibiting an inflammatoryresponse; iii) inhibiting macrophage activation; iii) activatingB-cells; iv) activating growth and survival of T-cells; v)downregulating C5a and C3a in monocytes and dendritic cells, vi)modulating activity of the IL-4 receptor is beneficial, said methodcomprising a step of administering a compound of the invention, antibodyof the invention or a pharmaceutical composition comprising said peptidesequence, said compound or said antibody to an individual in need.

DESCRIPTION OF DRAWINGS

FIG. 1.

Structure of IL-4 in complex with the ectodomain of IL-4Rα (PDB ID:1IAR). Location of peptide1 (SEQ ID NO:2) (left) and peptide3 (SEQ IDNO:3) (right) is indicated in grey.

FIG. 2.

Structure of IL-4 in complex with the ectodomain of IL-4Rα (PDB ID:1IAR). Location of peptide3a (SEQ ID NO:1) (left) and peptide4 (SEQ IDNO: 4) (right) is indicated in grey.

FIG. 3.

Structure of IL-4 in complex with the ectodomain of γc common receptor(PDB ID: 3BPL). Location of peptide1 (SEQ ID NO:2) (left) and peptide3(SEQ ID NO:3) (right) is indicated in grey.

FIG. 4

Structure of IL-4 in complex with the ectodomain of IL-4Rα and γc commonreceptor (PDB ID: 3BPL). Location of peptide3a (SEQ ID NO:1) (left) andpeptide4 (SEQ ID NO: 4) (right) is indicated in grey.

FIG. 5.

Effect of IL-4-derived peptide Ph1 (SEQ ID NO:2) on neurite outgrowth incultures of cerebellar granule neurons. The P2d peptide was used apositive control (see Soroka et al., 2002).

FIG. 6.

Effect of Ph2 (SEQ ID NO:3) on neurite outgrowth in cultures ofcerebellar granule neurons. Level of significance compared to control isrepresented as followed: ***=p<0.001. Seven independent experiments wereperformed.

FIG. 7.

Macrophage secretion of TNF-α when pre-treated with Ph2 (SEQ ID N0:3).

A: Column diagram of the amount of TNF-α released from macrophages whennot pre-treated with Ph2 or activated by IFN-γ (striped column), whenactivated with 0.01 μg/ml IFN-γ (white column) or when pre-treated with100 μM hydrocortisone and activated with 0.01 μg/ml IFN-γ (blackcolumn). Level of significance compared to TNF-α amount released fromnon-pre-treated, activated macrophages (white column) are represented asfollowed: ***=p<0.001. B: Column diagram of the amount of TNF-α releasedfrom macrophages when pre-treated with Ph2 in various concentrationsbefore activation with 0.01 μg/ml IFN-γ. Level of significance comparedto TNF-α amount released from non-pre-treated, activated macrophages (0column) is represented as followed: ***=p<0.001. Results in both figuresare shown as percentages of the untreated control, only activated byIFN-γ. Results from six independent experiments are shown for thecontrols and the Ph2 concentrations 9, 27, 81 and 243 μg/ml.

FIG. 8.

Binding of Ph2 (SEQ ID NO:3) to IL4rα.

Binding study by applying Surface Plasmon Resonance. A: As a control,binding between IL4 and IL4rα was investigated by immobilizing IL4rα ona chip and then IL4 was run over the chip in solution. B: Bindingbetween Ph2 and IL4rα was studied by immobilizing Ph2 on the chip andIL4rα was run over the chip in solution. Results were analysed and KDwas calulated with the computer software BIAevaluation.

FIG. 9.

Effect of Ph3 (SEQ ID NO:1) on neurite outgrowth in cultures ofcerebellar granule neurons. Level of significance compared to control isrepresented as followed: **=p<0.01. Seven independent experiments wereperformed.

FIG. 10.

Macrophage secretion of TNF-α when pre-treated with Ph3 (SEQ ID NO:1).

A: Column diagram of the amount of TNF-α released from macrophages whennot pre-treated with Ph3 or activated by IFN-γ (striped column), whenactivated with 0.01 μg/ml IFN-γ (white column) or when pre-treated with100 μM hydrocortisone and activated with 0.01 μg/ml IFN-γ (blackcolumn). Level of significance compared to TNF-α amount released fromnon-pre-treated, activated macrophages (white column) are represented asfollowed: ***=p<0.001. B: Column diagram of the amount of TNF-α releasedfrom macrophages when pre-treated with Ph3 in various concentrationsbefore activation with 0.01 μg/ml IFN-γ. Level of significance comparedto TNF-α amount released from non-pre-treated, activated macrophages (0column) is represented as followed: ***=p<0.001. Results in both figuresare shown as percentages of the untreated control, only activated byIFN-γ. Results from six independent experiments are shown for thecontrols and the Ph3 concentrations 9, 27, and 81 μg/ml.

FIG. 11.

Binding of Ph3 (SEQ ID NO:1) to IL4rα.

Binding study by applying Surface Plasmon Resonance. A: As a control,binding between IL4 and IL4rα was investigated by immobilizing IL4rα ona chip and then IL4 was run over the chip in solution. B: Bindingbetween Ph3 and IL4rα was studied by immobilizing Ph3 on the chip andIL4rα was run over the chip in solution. Results were analysed and KDwas calculated with the computer software BIAevaluation.

FIG. 12.

Effect of Ph4 (SEQ ID NO:4) on neurite outgrowth in cultures ofcerebellar granule neurons. Level of significance compared to control isrepresented as followed: *=p<0.05, **=p<0.01. Five independentexperiments were performed.

FIG. 13.

Macrophage secretion of TNF-α when pre-treated with Ph5 (SEQ ID NO:5).

A: Column diagram of the amount of TNF-α released from macrophages whennot pre-treated with Ph4 or activated by IFN-γ (stripes), when activatedwith 0.01 μg/ml IFN-γ (white) and when pre-treated with 100 μMhydrocortisone and activated with 0.01 μg/ml IFN-γ (black). B: Columndiagram of the amount of TNF-α released from macrophages whenpre-treated with 9 μg/ml Ph5 before activation with 0.01 pg/ml IFN-γ.Two independent experiments were performed.

FIG. 14.

Macrophage secretion of TNF-α when pre-treated with Ph6 (SEQ ID NO:6).

A: Column diagram of the amount of TNF-α released from macrophages whennot pre-treated with Ph4 or activated by IFN-γ (stripes), when activatedwith 0.01 μg/ml IFN-γ (white) and when pre-treated with 100 μMhydrocortisone and activated with 0.01 μg/ml IFN-γ (black). B: Columndiagram of the amount of TNF-α released from macrophages whenpre-treated with various concentrations of Ph6 before activation with0.01 μg/ml IFN-γ. Two independent experiments were performed.

FIG. 15.

Macrophage secretion of TNF-α when pre-treated with Ph8 (SEQ ID NO:1).

A: Column diagram of the amount of TNF-α released from macrophages whennot pre-treated with Ph3 or activated by IFN-γ (striped column), whenactivated with 0.01 μg/ml IFN-γ (white column) or when pre-treated with100 μM hydrocortisone and activated with 0.01 μg/ml IFN-γ (blackcolumn). Level of significance compared to TNF-α amount released fromnon-pre-treated, activated macrophages (white column) are represented asfollowed: ***=p<0.001. B: Column diagram of the amount of TNF-α releasedfrom macrophages when pre-treated with Ph8 in various concentrationsbefore activation with 0.01 μg/ml IFN-γ. Level of significance comparedto TNF-α amount released from non-pre-treated, activated macrophages (0column) is represented as followed: ***=p<0.001. Results in both figuresare shown as percentages of the untreated control, only activated byIFN-γ. Results from six independent experiments are shown for thecontrols and the Ph8 concentrations 9, 27, 81 and 243 μg/ml.

FIG. 16.

Macrophage secretion of TNF-α when pre-treated with Ph10 (SEQ ID:1).

A: Column diagram of the amount of TNF-α released from macrophages whennot pre-treated with Ph10 or activated by IFN-γ (striped column), whenactivated with 0.01 μg/ml IFN-γ (white column) or when pre-treated with100 μM hydrocortisone and activated with 0.01 μg/ml IFN-γ (blackcolumn). Level of significance compared to TNF-α amount released fromnon-pre-treated, activated macrophages (white column) are represented asfollowed: **=p<0.01. B: Column diagram of the amount of TNF-α releasedfrom macrophages when pre-treated with Ph10 in various concentrationsbefore activation with 0.01 μg/ml IFN-γ. Level of significance comparedto TNF-α amount released from non-pre-treated, activated macrophages (0column) is represented as followed: **=p<0.01. Results in both figuresare shown as percentages of the untreated control, only activated byIFN-γ. Results from four independent experiments are shown for thecontrols and the Ph10 concentrations 9, 27, 81 and 243 μg/ml. Only twoexperiments were performed with the concentration 54 μg/ml Ph10 whichdoes that these data were not included in the statistical analysis.

FIG. 17.

Macrophage secretion of TNF-α when pre-treated with Ph12 (SEQ ID:19).

A: Column diagram of the amount of TNF-α released from macrophages whennot pre-treated with Ph12 or activated by IFN-γ (stripes), whenactivated with 0.01 μg/ml IFN-γ (white) and when pre-treated with 100 μMhydrocortisone and activated with 0.01 μg/ml IFN-γ (black). B: Columndiagram of the amount of TNF-α released from macrophages whenpre-treated with Ph12 in various concentrations before activation with0.01 μg/ml IFN-γ. Two independent experiments were performed.

DETAILED DESCRIPTION OF THE INVENTION

A compound according to the invention can be a fragment derived frominterleukin-4, or it may be derived from a variant of interleukin-4,such as a natural or recombinant interleukin-4 variant, for example ainterleukin-4 variant produced by alternative splicing, or geneticpolymorphism, or any type of recombinant interleukin-4.

A peptide according to the invention is a peptide which is capable ofinteracting with the IL-4 receptor, modulating IL-4 receptor signalling,activating B-cells, activating growth and survival of T-cells,downregulating C5a and C3a in monocytes and dendritic cells orinhibiting macrophage activation.

By the terms “modulation” or “modulating” are meant a change, such as aninhibition or stimulation. By the term “interacting” is meant an action,such as binding, between the peptide and the IL-4 receptor which causean effect.

Amino Acid Sequence

Compounds according to the invention comprise a peptide consisting of acontiguous amino acid sequence derived from IL-4 or a fragment orvariant thereof.

In one embodiment the compound according to the invention may comprise apeptide consisting of at most 35 contiguous amino acids which is derivedfrom interleukin-4 (SEQ ID:38) or a fragment thereof, or a variant beingat least 70% identical to SEQ ID NO:38 or a fragment thereof.

The amino acid sequence of the human IL-4 precursor (Swiss-Prot ID:P05112) is:

(SEQ ID NO: 38) MGLTSQLLPP LFFLLACAGN FVHGHKCDIT LQEIIKTLNSLTEQKTLCTE LTVTDIFAAS KNTTEKETFC RAATVLRQFYSHHEKDTRCL GATAQQFHRH KQLIRFLKRL DRNLWGLAGLNSCPVKEANQ STLENFLERL KTIMREKYSK CSS

A peptide sequence according to the invention consists of at most 35contiguous amino acid residues, such as from 3 to 35 amino acidresidues, such as from 3 to 30, for example from 3 to 25, such as from 5to 25, such as form 7 to 25, such as from 8 to 25, for example from 10to 25, or from 12 to 25, such as from 14 to 25. Sequences comprisingfrom 5 to 25 contiguous amino acid residues are preferred.

In a preferred embodiment said peptides of the invention comprise atmost 35 contiguous amino acids which are derived from an alpha-helix ofIL-4.

By the term “alpha-helix” is meant the common motif in the secondarystructure of proteins, the alpha helix (α-helix) is a right- orleft-handed coiled conformation, in which every backbone N—H groupdonates a hydrogen bond to the backbone C═O group of the amino acid fourresidues earlier.

In a preferred embodiment said peptides of the invention comprise asequence with the formula

X1—X2—X3, wherein

X1 is L,

X2 is I, Q, G, T, or a charged amino acid; and

X3 is Q, T, or a charged amino acid.

In one preferred embodiment X2 is I or Q.

In a more preferred embodiment X2 is I.

In another more preferred embodiment X2 is Q.

In one preferred embodiment X2 is a charged amino acid.

In a preferred embodiment X3 is a charged amino acid.

In a more preferred embodiment X3 is R or E.

In one most preferred embodiment X3 is R.

In another more preferred embodiment X3 is E.

In another preferred embodiment X3 is Q or T.

In an even more preferred embodiment X1 is L, X2 is I, and X3 is R.

In another even more preferred embodiment X1 is L, X2 is Q and X3 is E.

In a most preferred embodiment said peptides of the invention consist ofan amino acid sequence selected from one of the following amino acidsequences:

AQFHRHKQLIRFLKRA SEQ ID NO: 1 AITLQEIIKTLNSA SEQ ID NO: 2 ARFLKRLDRNLWGGSEQ ID NO: 3 AERLKTIMREKYSKS SEQ ID NO: 4 LQEIKTLN SEQ ID NO: 5KRLQQNLFGG SEQ ID NO: 6 Ac-AQFHRHKQLIRFLKRA SEQ ID NO: 7 QEIIKKLSEQ ID NO: 8 AIQNQEEIKYLNS SEQ ID NO: 9 AIILQEI SEQ ID NO: 10 IVLQEII SEQ ID NO: 11 TLGEIIKGVNS SEQ ID NO: 12 VTLIDHSEEIFKTLN SEQ ID NO: 13LQERIKSLN SEQ ID NO: 14 RLDRENVAVYNLW SEQ ID NO: 15 LRSLDRNLSEQ ID NO: 16 RLLRLDRN SEQ ID NO: 17 RFLKRYFYNLEENL SEQ ID NO: 18RNKQVIDSLAKFLKR SEQ ID NO: 19 RHKALIR SEQ ID NO: 20 KKLIRYLKSEQ ID NO: 21 RHKTLIR SEQ ID NO: 22 MQDKYSKS SEQ ID NO: 23AERVKIEQREYKKYS SEQ ID NO: 24 SQLIRFLKRLA SEQ ID NO: 25 TVTDIFAASKNTTSEQ ID NO: 26 TLENFLERLKTA SEQ ID NO: 27 TEKEVLRQFYSA SEQ ID NO: 28KTLTELTKTLNS SEQ ID NO: 29 AHKEIIKTLNSLQKA SEQ ID NO: 30 AKTLSTELTVTASEQ ID NO: 31 STLENFLERLA SEQ ID NO: 32 NEERLKTIMRA SEQ ID NO: 33RAATVLRQFYSR SEQ ID NO: 34 KTLNSLTEQKT SEQ ID NO: 35 AHRHKQLIRASEQ ID NO: 36 ATAQQFHRHKQA SEQ ID NO: 37or a variant or fragment thereof.

In one embodiment the said peptides of the invention consist of an aminoacid sequence selected from one of the following amino acid sequences:

AQFHRHKQLIRFLKRA (SEQ ID NO: 1) Ac-AQFHRHKQLIRFLKRA (SEQ ID NO: 7)RHKALIR (SEQ ID NO: 20) KKLIRYLK (SEQ ID NO: 21) RHKTLIR (SEQ ID NO: 22)SQLIRFLKRLA (SEQ ID NO: 25) AHRHKQLIRA (SEQ ID NO: 36)or a variant or fragment thereof.

In one embodiment the said peptides of the invention consist of an aminoacid sequence selected from one of the following amino acid sequences:

AITLQEIIKTLNSA (SEQ ID NO: 2) LQEIKTLN (SEQ ID NO: 5) AIILQEI(SEQ ID NO: 10) IVLQEII (SEQ ID NO: 11) LQERIKSLN (SEQ ID NO: 14)AHKEIIKTLNSLQKA (SEQ ID NO: 30)or a variant or fragment thereof.

In the present context the standard one-letter code for amino acidresidues as well as the standard three-letter code are applied.Abbreviations for amino acids are in accordance with the recommendationsin the IUPAC-IUB Joint Commission on Biochemical Nomenclature Eur. J.Biochem, 1984, vol. 184, pp 9-37. Throughout the description and claimseither the three letter code or the one letter code for natural aminoacids are used. Where the L or D form has not been specified it is to beunderstood that the amino acid in question has the natural L form, cf.Pure & Appl. Chem. Vol. (56(5) pp 595-624 (1984) or the D form, so thatthe peptides formed may be constituted of amino acids of L form, D form,or a sequence of mixed L forms and D forms.

Where nothing is specified it is to be understood that the C-terminalamino acid of a peptide for use according to the invention exists as thefree carboxylic acid, this may also be specified as “—OH”. However, theC-terminal amino acid of a peptide for use according to the inventionmay be the amidated derivative, which is indicated as “—NH₂”. Wherenothing else is stated the N-terminal amino acid of a polypeptidecomprises a free amino-group, this may also be specified as “H—”.

A peptide, fragment or variant thereof according to the invention canalso comprise one or several unnatural amino acids.

A preferred peptide according to the invention is an isolated contiguouspeptide sequence which comprises at most 35 amino acid residues of IL-4.It is understood that all peptides according to the invention compriseat least one amino acid sequence selected from any of the sequences SEQID NOs: 1-37 or a fragment or variant thereof.

Thus, some embodiments of the invention may relate to a peptidecomprising a fragment of a sequence selected from SEQ ID NOs:1 to 37.Another embodiment may relate to variants of SEQ ID NOs:1-37.

In one embodiment a variant fragment varies compared to a fragment ofSEQ ID NO 38. A variant fragment may differ from a fragment of SEQ ID NO38 by having a different amino acid at one or more positions. Preferablythe variant differs from the fragment of SEQ ID NO 38 at up to 10 aminoacid positions, more preferably at up to 8 position, such as up to 6positions, for example up to 5 positions, such as at 4, 3, 2 or 1position. Such variants may also differ from a fragment of SEQ ID NO 38in other ways, such as by having one or more chemical modifications.

A variant according to the invention of an amino acid sequence selectedfrom the sequences SEQ ID NOs: 1-38 may be

-   -   i) an amino acid sequence which has at least 70% identity with a        selected sequence, such as 71-75% identity, for example 76-80%        identity, such as 81-85% identity, such as 86-90% identity, for        example 91-95% identity, such as 96-99% identity, wherein the        identity is defined as a percentage of identical amino acids in        said sequence when it is collated with the selected sequence.        The identity between amino acid sequences may be calculated        using well known algorithms such as BLOSUM 30, BLOSUM 40, BLOSUM        45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65,        BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, or BLOSUM 90;    -   ii) an amino acid sequence which has at least 70% positive amino        acid matches with a selected sequence, such as 71-80% positive        amino acid matches, for example 81-85% positive amino acid        matches, such as 86-90% positive amino acid matches, for example        91-95% positive amino acid matches, such as 96-99% positive        amino acid matches, wherein the positive amino acid match is        defined as the presence at the same position in two compared        sequences of amino acid residues which has similar physical        and/or chemical properties. Preferred positive amino acid        matches of the present invention are K to R, E to D, L to M, Q        to E, I to V, I to L, A to S, Y to W, K to Q, S to T, N to S and        Q to R;    -   iii) an amino acid sequence which is identical to a selected        sequence, or it has at least 70% identity with said sequence        such as 71-80% identity, for example 81-85% identity, such as        86-90% identity, for example 91-95% identity, such as 96-99%        identity, or has at least 75% positive amino acid matches with        the selected sequence, such as 76-80% positive amino acid        matches, for example 81-85% positive amino acid matches, such as        86-90% positive amino acid matches, for example 91-95% positive        amino acid matches, such as 96-99% positive amino acid matches,        and comprises other chemical moieties, e. g. phosphoryl,        sulphur, acetyl, glycosyl moieties.

The term “variant of a peptide sequence” also means that the peptidesequence may be modified, for example by substitution of one or more ofthe amino acid residues. Both L-amino acids and D-amino acids may beused. Other modification may comprise derivatives such as esters,sugars, etc., for example methyl and acetyl esters, as well aspolyethylene glycol modifications.

Furthermore, an amine group of the peptide may be converted to amides,wherein the acid part of the amide is a fatty acid.

In another aspect, variants of the amino acid sequences according to theinvention may comprise, within the same variant, or fragments thereof oramong different variants, or fragments thereof, at least onesubstitution, such as a plurality of substitutions introducedindependently of one another. Variants of the complex, or fragmentsthereof may thus comprise conservative substitutions independently ofone another, wherein at least one glycine (Gly) of said variant, orfragments thereof is substituted with an amino acid selected from thegroup of amino acids consisting of Ala, Val, Leu, and Ile, andindependently thereof, variants, or fragments thereof, wherein at leastone alanine (Ala) of said variants, or fragments thereof is substitutedwith an amino acid selected from the group of amino acids consisting ofGly, Val, Leu, and Ile, and independently thereof, variants, orfragments thereof, wherein at least one valine (Val) of said variant, orfragments thereof is substituted with an amino acid selected from thegroup of amino acids consisting of Gly, Ala, Leu, and Ile, andindependently thereof, variants, or fragments thereof, wherein at leastone leucine (Leu) of said variant, or fragments thereof is substitutedwith an amino acid selected from the group of amino acids consisting ofGly, Ala, Val, and Ile, and independently thereof, variants, orfragments thereof, wherein at least one isoleucine (Ile) of saidvariants, or fragments thereof is substituted with an amino acidselected from the group of amino acids consisting of Gly, Ala, Val andLeu, and independently thereof, variants, or fragments thereof whereinat least one aspartic acids (Asp) of said variant, or fragments thereofis substituted with an amino acid selected from the group of amino acidsconsisting of Glu, Asn, and Gin, and independently thereof, variants, orfragments thereof, wherein at least one aspargine (Asn) of saidvariants, or fragments thereof is substituted with an amino acidselected from the group of amino acids consisting of Asp, Glu, and Gin,and independently thereof, variants, or fragments thereof, wherein atleast one glutamine (Gln) of said variants, or fragments thereof issubstituted with an amino acid selected from the group of amino acidsconsisting of Asp, Glu, and Asn, and wherein at least one phenylalanine(Phe) of said variants, or fragments thereof is substituted with anamino acid selected from the group of amino acids consisting of Tyr,Trp, His, Pro, and preferably selected from the group of amino acidsconsisting of Tyr and Trp, and independently thereof, variants, orfragments thereof, wherein at least one tyrosine (Tyr) of said variants,or fragments thereof is substituted with an amino acid selected from thegroup of amino acids consisting of Phe, Trp, His, Pro, preferably anamino acid selected from the group of amino acids consisting of Phe andTrp, and independently thereof, variants, or fragments thereof, whereinat least one arginine (Arg) of said fragment is substituted with anamino acid selected from the group of amino acids consisting of Lys andHis, and independently thereof, variants, or fragments thereof, whereinat least one lysine (Lys) of said variants, or fragments thereof issubstituted with an amino acid selected from the group of amino acidsconsisting of Arg and His, and independently thereof, variants, orfragments thereof, and independently thereof, variants, or fragmentsthereof, and wherein at least one proline (Pro) of said variants, orfragments thereof is substituted with an amino acid selected from thegroup of amino acids consisting of Phe, Tyr, Trp, and His, andindependently thereof, variants, or fragments thereof, wherein at leastone cysteine (Cys) of said variants, or fragments thereof is substitutedwith an amino acid selected from the group of amino acids consisting ofAsp, Glu, Lys, Arg, His, Asn, Gln, Ser, Thr, and Tyr.

It thus follows from the above that the same variant of a peptidefragment, or fragment of said variant may comprise more than oneconservative amino acid substitution from more than one group ofconservative amino acids as defined herein above. The term “conservativeamino acid substitution” is used synonymously herein with the term“homologous amino acid substitution”.

The groups of conservative amino acids are as the following:

A, G (neutral, weakly hydrophobic),

Q, N, S, T (hydrophilic, non-charged)

E, D (hydrophilic, acidic)

H, K, R (hydrophilic, basic)

L, P, I, V, M, F, Y, W (hydrophobic, aromatic)

C (cross-link forming)

Conservative substitutions may be introduced in any position of apreferred predetermined peptide for use according to the invention orfragment thereof. It may however also be desirable to introducenon-conservative substitutions, particularly, but not limited to, anon-conservative substitution in any one or more positions.

A non-conservative substitution leading to the formation of a variantfragment of the peptide for use according to the invention would forexample differ substantially in polarity, for example a residue with anon-polar side chain (Ala, Leu, Pro, Trp, Val, Ile, Leu, Phe or Met)substituted for a residue with a polar side chain such as Gly, Ser, Thr,Cys, Tyr, Asn, or Gln or a charged amino acid such as Asp, Glu, Arg, orLys, or substituting a charged or a polar residue for a non-polar one;and/or ii) differ substantially in its effect on peptide backboneorientation such as substitution of or for Pro or Gly by anotherresidue; and/or iii) differ substantially in electric charge, forexample substitution of a negatively charged residue such as Glu or Aspfor a positively charged residue such as Lys, His or Arg (and viceversa); and/or iv) differ substantially in steric bulk, for examplesubstitution of a bulky residue such as His, Trp, Phe or Tyr for onehaving a minor side chain, e.g. Ala, Gly or Ser (and vice versa).

Substitution of amino acids may in one embodiment be made based upontheir hydrophobicity and hydrophilicity values and the relativesimilarity of the amino acid side-chain substituents, including charge,size, and the like.

A peptide according to the invention is a peptide which is capable ofinteracting with the IL-4 receptor.

In one embodiment the peptide according to the invention is capable ofmodulating IL-4 receptor signalling.

In a preferred embodiment the peptide according to the invention iscapable of stimulating IL-4 signalling. In another preferred embodimentthe peptide according to the invention is capable of inhibiting IL-4receptor signalling.

In another embodiment the peptide according to the invention is capableof activating B-cells.

In a further embodiment the peptide according to the invention iscapable of activating growth and survival of T-cells.

In another embodiment the peptide according to the invention is capableof downregulating C5a and C3a in monocytes and dendritic cells.

In yet another embodiment the peptide according to the invention iscapable of inhibiting macrophage activation.

Both fragments and variants of amino acid sequences according to theinvention are functional equivalents of said sequences.

By the term “functional equivalent” of an amino acid sequence is in thepresent context meant a molecule which meets the criteria for a variantor a fragment of said amino acid sequence described above and which iscapable of one or more functional activities of said sequence or acompound comprising said sequence. In a preferred embodiment, thefunctional equivalent of an amino acid sequence according to theinvention, is capable of interacting with the IL-4 receptor and modulateIL-4 receptor signalling.

The invention relates both to isolated peptides according to theinvention and fusion proteins comprising peptides according to theinvention.

In one embodiment, the peptide according to the invention is an isolatedpeptide. By the term “isolated peptide” is meant that the peptideaccording to the invention is an individual compound and not a part ofanother compound. The isolated peptide may be produced by use of anyrecombinant technology methods or chemical synthesis and separated fromother compounds, or it may be separated from a longer polypeptide orprotein by a method of enzymatic or chemical cleavage and furtherseparated from other protein fragments.

The peptide sequence may be present in the compound as a single copy,i.e. formulated as a monomer of the peptide sequence, or it may bepresent as several copies of the same sequence, e.g. as a multimercomprising two or more copies of a sequence selected from SEQ IDNOs:1-37, or two or more copies of a fragment or a variant of saidsequence.

An isolated peptide according to the invention may in another embodimentcomprise a fragment of interleukin-4 which consists of a contiguousamino acid sequence derived from interleukin-4, selected from SEQ IDNOs:1-37 or a variant thereof. In another embodiment the isolatedpeptide may consist of one or more of the sequences SEQ ID NOs:1-37.

Production of Peptide Sequences

The peptide sequences of the present invention may be prepared by anyconventional synthetic methods, recombinant DNA technologies, enzymaticcleavage of full-length proteins which the peptide sequences are derivedfrom, or a combination of said methods.

Synthetic Preparation

The methods for synthetic production of peptides are well known in theart. Detailed descriptions as well as practical advice for producingsynthetic peptides may be found in Synthetic Peptides: A User's Guide(Advances in Molecular Biology), Grant G. A. ed., Oxford UniversityPress, 2002, or in: Pharmaceutical Formulation: Development of Peptidesand Proteins, Frokjaer and Hovgaard eds., Taylor and Francis, 1999.

Peptides may for example be synthesised by using Fmoc chemistry and withAcm-protected cysteins. After purification by reversed phase HPLC,peptides may be further processed to obtain for example cyclic or C- orN-terminal modified isoforms. The methods for cyclization and terminalmodification are well-known in the art and described in detail in theabove-cited manuals.

In a preferred embodiment the peptide sequences of the invention areproduced synthetically, in particular, by the Sequence Assisted PeptideSynthesis (SAPS) method.

Peptides may be synthesised either batchwise in a polyethylene vesselequipped with a polypropylene filter for filtration or in thecontinuous-flow version of the polyamide solid-phase method (Dryland, A.and Sheppard, R. C., (1986) J. Chem. Soc. Perkin Trans. I, 125-137.) ona fully automated peptide synthesiser using 9-fluorenylmethyloxycarbonyl(Fmoc) or tent. -Butyloxycarbonyl, (Boc) as N-a-amino protecting groupand suitable common protection groups for side-chain functionality's.

Recombinant Preparation

Thus, in one embodiment the peptides of the invention are produced byuse of recombinant DNA technologies.

The DNA sequence encoding a peptide or the corresponding full-lengthprotein the peptide originates from may be prepared synthetically byestablished standard methods, e.g. the phosphoamidine method describedby Beaucage and Caruthers, 1981, Tetrahedron Lett. 22:1859-1869, or themethod described by Matthes et al., 1984, EMBO J. 3:801-805. Accordingto the phosphoamidine method, oligonucleotides are synthesised, e.g. inan automatic DNA synthesiser, purified, annealed, ligated and cloned insuitable vectors.

The DNA sequence encoding a peptide may also be prepared byfragmentation of the

DNA sequences encoding the corresponding full-length protein of peptideorigin, using DNAase I according to a standard protocol (Sambrook etal., Molecular cloning: A Laboratory manual. 2 rd ed., CSHL Press, ColdSpring Harbor, N.Y., 1989). The present invention relates to full-lengthproteins selected from the groups of proteins identified above. The DNAencoding the full-length proteins of the invention may alternatively befragmented using specific restriction endonucleases. The fragments ofDNA are further purified using standard procedures described in Sambrooket al., Molecular cloning: A Laboratory manual. 2 rd ed., CSHL Press,Cold Spring Harbor, N.Y., 1989.

The DNA sequence encoding a full-length protein may also be of genomicor cDNA origin, for instance obtained by preparing a genomic or cDNAlibrary and screening for DNA sequences coding for all or part of thefull-length protein by hybridisation using synthetic oligonucleotideprobes in accordance with standard techniques (cf. Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989). The DNA sequence may also be prepared by polymerase chainreaction using specific primers, for instance as described in U.S. Pat.No. 4,683,202 or Saiki et al., 1988, Science 239:487-491.

The DNA sequence is then inserted into a recombinant expression vector,which may be any vector, which may conveniently be subjected torecombinant DNA procedures. The choice of vector will often depend onthe host cell into which it is to be introduced. Thus, the vector may bean autonomously replicating vector, i.e. a vector that exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome and replicated together with the chromosome(s) intowhich it has been integrated.

In the vector, the DNA sequence encoding a peptide or a full-lengthprotein should be operably connected to a suitable promoter sequence.The promoter may be any DNA sequence, which shows transcriptionalactivity in the host cell of choice and may be derived from genesencoding proteins either homologous or heterologous to the host cell.Examples of suitable promoters for directing the transcription of thecoding DNA sequence in mammalian cells are the SV 40 promoter (Subramaniet al., 1981, Mol. Cell Biol. 1:854-864), the MT-1 (metallothioneingene) promoter (Palmiter et al., 1983, Science 222: 809-814) or theadenovirus 2 major late promoter. A suitable promoter for use in insectcells is the polyhedrin promoter (Vasuvedan et al., 1992, FEBS Lett.311:7-11). Suitable promoters for use in yeast host cells includepromoters from yeast glycolytic genes (Hitzeman et al., 1980, J. Biol.Chem. 255:12073-12080; Alber and Kawasaki, 1982, J. Mol. Appl. Gen. 1:419-434) or alcohol dehydrogenase genes (Young et al., 1982, in GeneticEngineering of Microorganisms for Chemicals, Hollaender et al, eds.,Plenum Press, New York), or the TPI1 (U.S. Pat. No. 4,599,311) orADH2-4c (Russell et al., 1983, Nature 304:652-654) promoters. Suitablepromoters for use in filamentous fungus host cells are, for instance,the ADH3 promoter (McKnight et al., 1985, EMBO J. 4:2093-2099) or thetpiA promoter.

The coding DNA sequence may also be operably connected to a suitableterminator, such as the human growth hormone terminator (Palmiter etal., op. cit.) or (for fungal hosts) the TPI1 (Alber and Kawasaki, op.cit.) or ADH3 (McKnight et al., op. cit.) promoters. The vector mayfurther comprise elements such as polyadenylation signals (e.g. from SV40 or the adenovirus 5 Elb region), transcriptional enhancer sequences(e.g. the SV 40 enhancer) and translational enhancer sequences (e.g. theones encoding adenovirus VA RNAs).

The recombinant expression vector may further comprise a DNA sequenceenabling the vector to replicate in the host cell in question. Anexample of such a sequence (when the host cell is a mammalian cell) isthe SV 40 origin of replication. The vector may also comprise aselectable marker, e.g. a gene the product of which complements a defectin the host cell, such as the gene coding for dihydrofolate reductase(DHFR) or one which confers resistance to a drug, e.g. neomycin,hydromycin or methotrexate.

The procedures used to ligate the DNA sequences coding the peptides orfull-length proteins, the promoter and the terminator, respectively, andto insert them into suitable vectors containing the informationnecessary for replication, are well known to persons skilled in the art(cf., for instance, Sambrook et al., op.cit.).

To obtain recombinant peptides of the invention the coding DNA sequencesmay be usefully fused with a second peptide coding sequence and aprotease cleavage site coding sequence, giving a DNA construct encodingthe fusion protein, wherein the protease cleavage site coding sequencepositioned between the HBP fragment and second peptide coding DNA,inserted into a recombinant expression vector, and expressed inrecombinant host cells. In one embodiment, said second peptide selectedfrom, but not limited by the group comprising glutathion-S-reductase,calf thymosin, bacterial thioredoxin or human ubiquitin natural orsynthetic variants, or peptides thereof. In another embodiment, apeptide sequence comprising a protease cleavage site may be the FactorXa, with the amino acid sequence IEGR, enterokinase, with the amino acidsequence DDDDK, thrombin, with the amino acid sequence LVPR/GS, orAcharombacter lyticus, with the amino acid sequence XKX, cleavage site.

The host cell into which the expression vector is introduced may be anycell which is capable of expression of the peptides or full-lengthproteins, and is preferably a eukaryotic cell, such as invertebrate(insect) cells or vertebrate cells, e.g. Xenopus laevis oocytes ormammalian cells, in particular insect and mammalian cells. Examples ofsuitable mammalian cell lines are the HEK293 (ATCC CRL-1573), COS (ATCCCRL-1650), BHK (ATCC CRL-1632, ATCC CCL-10) or CHO (ATCC CCL-61) celllines. Methods of transfecting mammalian cells and expressing DNAsequences introduced in the cells are described in e.g. Kaufman andSharp, J. Mol. Biol. 159, 1982, pp. 601-621; Southern and Berg, 1982, J.Mol. Appl. Genet. 1:327-341; Loyter et al., 1982, Proc. Natl. Acad. Sci.USA 79: 422-426; Wigler et al., 1978, Cell 14:725; Corsaro and Pearson,1981, in Somatic Cell Genetics 7, p. 603; Graham and van der Eb, 1973,Virol. 52:456; and Neumann et al., 1982, EMBO J. 1:841-845.

Alternatively, fungal cells (including yeast cells) may be used as hostcells. Examples of suitable yeast cells include cells of Saccharomycesspp. or Schizosaccharomyces spp., in particular strains of Saccharomycescerevisiae. Examples of other fungal cells are cells of filamentousfungi, e.g. Aspergillus spp. or Neurospora spp., in particular strainsof Aspergillus oryzae or Aspergillus niger. The use of Aspergillus spp.for the expression of proteins is described in, e.g., EP 238 023.

The medium used to culture the cells may be any conventional mediumsuitable for growing mammalian cells, such as a serum-containing orserum-free medium containing appropriate supplements, or a suitablemedium for growing insect, yeast or fungal cells. Suitable media areavailable from commercial suppliers or may be prepared according topublished recipes (e.g. in catalogues of the American Type CultureCollection).

The peptides or full-length proteins recombinantly produced by the cellsmay then be recovered from the culture medium by conventional proceduresincluding separating the host cells from the medium by centrifugation orfiltration, precipitating the proteinaceous components of thesupernatant or filtrate by means of a salt, e.g. ammonium sulphate,purification by a variety of chromatographic procedures, e.g. HPLC, ionexchange chromatography, affinity chromatography, or the like.

Medicament

It is an objective of the invention to provide a compound capable ofmodulating the activity of IL-4, said compound according to theinvention can be used as a medicament for the treatment of diseases,wherein modulation of IL-4 signalling may be considered as an essentialcondition for curing.

Accordingly, the invention relates to the use of one or more of thepeptides comprising a sequence derived from IL-4 or a fragment orvariant thereof for the manufacture of a medicament.

In one embodiment the medicament of the invention comprises at least oneof the amino acid sequences set forth in SEQ ID NOS: 1-37 or fragmentsor variants of said sequences. In another embodiment the medicament ofthe invention comprises an antibody capable of binding to an epitope inIL-4 or a fragment thereof or a fragment or variant of said antibody.

The medicament of the invention comprises an effective amount of one ormore of the compounds as defined above, or a composition comprising acompound as defined above, in combination with pharmaceuticallyacceptable additives. Such medicament may suitably be formulated fororal, percutaneous, subcutaneous, topical, intramuscular, intravenous,intracranial, intrathecal, intracerebroventricular, nasal, intranasal orpulmonal administration or parental administration supplemented withintraarticular administration into or near joint capsules.

Strategies in formulation development of medicaments and compositionsbased on the peptides of the present invention generally correspond toformulation strategies for any other protein-based drug product.Potential problems and the guidance required to overcome these problemsare dealt with in several textbooks, e.g. “Therapeutic Peptides andProtein Formulation. Processing and Delivery Systems”, Ed. A. K. Banga,Technomic Publishing AG, Basel, 1995.

Injectables are usually prepared either as liquid solutions orsuspensions, solid forms suitable for solution in, or suspension in,liquid prior to injection. The preparation may also be emulsified. Theactive ingredient is often mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol or the like, and combinations thereof. In addition, if desired,the preparation may contain minor amounts of auxiliary substances suchas wetting or emulsifying agents, pH buffering agents, or which enhancethe effectiveness or transportation of the preparation.

Formulations of the compounds of the invention can be prepared bytechniques known to the person skilled in the art. The formulations maycontain pharmaceutically acceptable carriers and excipients includingmicrospheres, liposomes, microcapsules, nanoparticles or the like.

The preparation may suitably be administered by injection, optionally atthe site, where the active ingredient is to exert its effect. Additionalformulations which are suitable for other modes of administrationinclude suppositories, nasal, pulmonal and, in some cases, oralformulations. For suppositories, traditional binders and carriersinclude polyalkylene glycols or triglycerides. Such suppositories may beformed from mixtures containing the active ingredient(s) in the range offrom 0.5% to 10%, preferably 1-2%. Oral formulations include suchnormally employed excipients as, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, and the like. These compositions takethe form of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations or powders and generally contain 10-95% of theactive ingredient(s), preferably 25-70%.

Other formulations are such suitable for nasal and pulmonaladministration, e.g. inhalators and aerosols.

The active compound may be formulated as neutral or salt forms.

Pharmaceutically acceptable salts include acid addition salts (forexample formed with the free amino groups of the peptide compound) andwhich are formed with inorganic acids such as, for example,hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitricacids and the like, or such organic acids as formic, acetic,trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric,fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic,picric, pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic,tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic,gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic,p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids andthe like. Salts formed with the free carboxyl group may also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

Further examples of pharmaceutically acceptable inorganic or organicacid addition salts include the pharmaceutically acceptable salts listedin J. Pharm. Sci. 1977, 66, 2, which is incorporated herein byreference. Examples of metal salts include lithium, sodium, potassium,magnesium salts and the like. Examples of ammonium and alkylatedammonium salts include ammonium, methylammonium, dimethylammonium,trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium,butylammonium, tetramethylammonium salts and the like.

The preparations are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective.The quantity to be administered depends on the subject to be treated,including, e.g. the weight and age of the subject, the disease to betreated and the stage of disease. Suitable dosage ranges are per kilobody weight normally of the order of several hundred pg activeingredient per administration with a preferred range of from about 0.1μg to 5000 μg per kilo body weight. Using monomeric forms of thecompounds, the suitable dosages are often in the range of from 0.1 μg to5000 μg per kilo body weight, such as in the range of from about 0.1 μgto 3000 μg per kilo body weight, and especially in the range of fromabout 0.1 μg to 1000 μg per kilo body weight. Using multimeric forms ofthe compounds, the suitable dosages are often in the range of from 0.1μg to 1000 μg per kilo body weight, such as in the range of from about0.1 μg to 750 μg per kilo body weight, and especially in the range offrom about 0.1 μg to 500 μg per kilo body weight such as in the range offrom about 0.1 μg to 250 μg per kilo body weight. In particular whenadministering nasally smaller dosages are used than when administeringby other routes. Administration may be performed once or may be followedby subsequent administrations. The dosage will also depend on the routeof administration and will vary with the age and weight of the subjectto be treated. A preferred dosage of multimeric forms would be in theinterval 1 mg to 70 mg per 70 kg body weight.

For most indications a localised or substantially localised applicationis preferred.

Some of the compounds of the present invention are sufficiently active,but for some of the others, the effect will be enhanced if thepreparation further comprises pharmaceutically acceptable additivesand/or carriers. Such additives and carriers will be known in the art.In some cases, it will be advantageous to include a compound, whichpromotes delivery of the active substance to its target.

In many instances, it will be necessary to administrate the formulationmultiple times. Administration may be a continuous infusion, such asintraventricular infusion or administration in more doses such as moretimes a day, daily, more times a week, weekly, etc. It is preferred thatadministration of the medicament is initiated before or shortly afterthe individual has been subjected to the factor(s) that may lead to celldeath. Preferably the medicament is administered within 8 hours from thefactor onset, such as within 5 hours from the factor onset. Many of thecompounds exhibit a long term effect whereby administration of thecompounds may be conducted with long intervals, such as 1 week or 2weeks.

In connection with the use in nerve guides, the administration may becontinuous or in small portions based upon controlled release of theactive compound(s). Furthermore, precursors may be used to control therate of release and/or site of release. Other kinds of implants and wellas oral administration may similarly be based upon controlled releaseand/or the use of precursors.

As discussed above, the present invention relates to treatment ofindividuals for inducing differentiation, modulating proliferation,stimulate regeneration, neuronal plasticity and survival of cells invitro or in vivo, the treatment involving administering an effectiveamount of one or more compounds as defined above.

Another strategy for administration is to implant or inject cellscapable of expressing and secreting the compound in question. Therebythe compound may be produced at the location where it is going to act.

Treatment

The compounds according to the invention are particularly useful fortreating inflammatory diseases and conditions. The compounds are usefulfor the diseases and conditions mentioned below, in particular usefulfor the treatment of inflammation in association with Rheumatoidarthritis and autoimmune diseases, as well as with Alzheimer's disease,Parkinson's disease and Huntington's disease.

Examples of disorders associated with inflammation that can be treatedwith the compounds of the invention include; neuroinflammation,Alzheimer's disease, Parkinson's disease and Huntington's disease,asthma and other allergic reactions, autoimmune diseases such as Acutedisseminated encephalomyelitis (ADEM), Addison's disease, ALS,Ankylosing spondylitis, Antiphospholipid antibody syndrome (APS),Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune inner eardisease, Bullous pemphigoid, Coeliac disease, Chagas disease, Chronicobstructive pulmonary disease, Dermatomyositis, Diabetes mellitus type1, Endometriosis, Goodpasture's syndrome, Graves' disease,Guillain-Barré syndrome (GBS), Hashimoto's disease, Hidradenitissuppurativa, Idiopathic thrombocytopenic purpura, Interstitial cystitis,Lupus erythematosus, Morphea, Multiple sclerosis, Myasthenia gravis,Narcolepsy, Neuromyotonia, Pemphigus Vulgaris, Pernicious anaemia,Polymyositis, Primary biliary cirrhosis, Rheumatoid arthritis,Schizophrenia, Scleroderma, Sjögren's syndrome, SLE, Temporal arteritis(also known as “giant cell arteritis”), Vasculitis, Vitiligo, Wegener'sgranulomatosis; chronic inflammation, chronic prostatitis,glomerulonephritis, hypersensitivities, inflammatory bowel diseases,pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis,transplant rejection, vasculitis, osteoarthritis, tendovaginitis, andarthritis.

The treatment may also be of persistent acute inflammation due tonon-degradable pathogens, persistent foreign bodies, or autoimmunereactions, inflammatory disease of the central nervous system, such asmeningitis, encephalitis, inflammatory and toxic neuropathy, includingacute infective polyneuritis, inflammatory disorders with tissue damage,HIV, hepatitis, osteoarthritis, tendovaginitis, and arthritis.

In one embodiment the treatment may be of non-immune diseases withaetiological origins in inflammatory processes including cancer,atherosclerosis, and ischaemic heart disease.

Antibody

It is an objective of the present invention to provide the use of anantibody, antigen binding fragment or recombinant protein thereofcapable of selectively binding to an epitope comprising a contiguousamino acid sequence derived from interleukin-4 or a fragment, homologueor variant thereof. The invention relates to any antibody capable ofselectively binding to an epitope comprising a contiguous amino acidsequence derived from interleukin-4, selected from any of the sequencesset forth in SEQ ID NOS: 1-37, or a fragment or variant of saidsequence.

By the term “epitope” is meant the specific group of atoms (on anantigen molecule) that is recognized by (that antigen's) antibodies. Theterm “epitope” is the equivalent to the term “antigenic determinant”.The epitope may comprise 3 or more amino acid residues, such as forexample 4, 5, 6, 7, 8 amino acid residues, located in close proximity,such as within a contiguous amino acid sequence, or located in distantparts of the amino acid sequence of an antigen, but due to proteinfolding have been approached to each other.

Antibody molecules belong to a family of plasma proteins calledimmunoglobulins, whose basic building block, the immunoglobulin fold ordomain, is used in various forms in many molecules of the immune systemand other biological recognition systems. A typical immunoglobulin hasfour polypeptide chains, containing an antigen binding region known as avariable region and a non-varying region known as the constant region.

Native antibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (VH) followed by a number of constant domains. Eachlight chain has a variable domain at one end (VL) and a constant domainat its other end. The constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light and heavy chain variable domains (Novotny J, & Haber E. ProcNatl Acad Sci USA. 82(14):4592-6, 1985).

Depending on the amino acid sequences of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are at least five (5) major classes of immunoglobulins: IgA, IgD,IgE, IgG and IgM, and several of these may be further divided intosubclasses (isotypes), e.g. IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 andIgA-2. The heavy chains constant domains that correspond to thedifferent classes of immunoglobulins are called alpha (α), delta (δ),epsilon (ε), gamma (γ) and mu (μ), respectively. The light chains ofantibodies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino sequences of their constantdomain. The subunit structures and three-dimensional configurations ofdifferent classes of immunoglobulins are well known.

The term “variable” in the context of variable domain of antibodies,refers to the fact that certain portions of the variable domains differextensively in sequence among antibodies. The variable domains are forbinding and determine the specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in three segments called complementarity determiningregions (CDRs) also known as hypervariable regions both in the lightchain and the heavy chain variable domains.

The more highly conserved portions of variable domains are called theframework (FR). The variable domains of native heavy and light chainseach comprise four FR regions, largely adopting a β-sheet configuration,connected by three CDRs, which form loops connecting, and in some casesforming part of, the β-sheet structure. The CDRs in each chain are heldtogether in close proximity by the FR regions and, with the CDRs fromthe other chain, contribute to the formation of the antigen-binding siteof antibodies. The constant domains are not involved directly in bindingan antibody to an antigen, but exhibit various effector functions, suchas participation of the antibody in antibody-dependent cellulartoxicity.

An antibody that is contemplated for use in the present invention thuscan be in any of a variety of forms, including a whole immunoglobulin,an antibody fragment such as Fv,

Fab, and similar fragments, a single chain antibody which includes thevariable domain complementarity determining regions (CDR), and the likeforms, all of which fall under the broad term “antibody”, as usedherein. The present invention contemplates the use of any specificity ofan antibody, polyclonal or monoclonal, and is not limited to antibodiesthat recognize and immunoreact with a specific antigen. In the contextof both the therapeutic and screening methods described below, preferredembodiments are the use of an antibody or fragment thereof that isimmunospecific for an antigen or epitope of the invention.

The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the antigen binding or variable region. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂ and Fv fragments. Papaindigestion of antibodies produces two identical antigen bindingfragments, called the Fab fragment, each with a single antigen bindingsite, and a residual “Fc” fragment, so-called for its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen binding fragments that are capable of cross-linkingantigen, and a residual other fragment (which is termed pFc′).Additional fragments can include diabodies, linear antibodies,single-chain antibody molecules, and multispecific antibodies formedfrom antibody fragments. As used herein, “functional fragment” withrespect to antibodies, refers to Fv, F(ab) and F(ab′)₂ fragments.

The term “antibody fragment” is used herein interchangeably with theterm “antigen binding fragment”.

Antibody fragments may be as small as about 4 amino acids, 5 aminoacids, 6 amino acids, 7 amino acids, 9 amino acids, about 12 aminoacids, about 15 amino acids, about 17 amino acids, about 18 amino acids,about 20 amino acids, about 25 amino acids, about 30 amino acids ormore. In general, an antibody fragment of the invention can have anyupper size limit so long as it is has similar or immunologicalproperties relative to antibody that binds with specificity to anepitope comprising a peptide sequence selected from any of the sequencesidentified herein as SEQ ID NOs: 1-37, or a fragment of said sequences.Thus, in context of the present invention the term “antibody fragment”is identical to the term “antigen binding fragment”.

Antibody fragments retain some ability to selectively bind with itsantigen or receptor. Some types of antibody fragments are defined asfollows:

-   -   -   (1) Fab is the fragment that contains a monovalent            antigen-binding fragment of an antibody molecule. A Fab            fragment can be produced by digestion of whole antibody with            the enzyme papain to yield an intact light chain and a            portion of one heavy chain.

    -   (2) Fab′ is the fragment of an antibody molecule can be obtained        by treating whole antibody with pepsin, followed by reduction,        to yield an intact light chain and a portion of the heavy chain.        Two Fab′ fragments are obtained per antibody molecule.

Fab′ fragments differ from Fab fragments by the addition of a fewresidues at the carboxyl terminus of the heavy chain CH1 domainincluding one or more cysteines from the antibody hinge region.

-   -   (3) (Fab′)₂ is the fragment of an antibody that can be obtained        by treating whole antibody with the enzyme pepsin without        subsequent reduction.    -   (4) F(ab′)₂ is a dimer of two Fab' fragments held together by        two disulfide bonds.

Fv is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy and one light chain variable domain in a tight, non-covalentassociation (V_(H)-V_(L) dimer). It is in this configuration that thethree CDRs of each variable domain interact to define an antigen bindingsite on the surface of the V_(H)-V_(L) dimer. Collectively, the six CDRsconfer antigen binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen,although at a lower affinity than the entire binding site.

-   -   (5) Single chain antibody (“SCA”), defined as a genetically        engineered molecule containing the variable region of the light        chain, the variable region of the heavy chain, linked by a        suitable polypeptide linker as a genetically fused single chain        molecule. Such single chain antibodies are also referred to as        “single-chain Fv” or “sFv” antibody fragments. Generally, the Fv        polypeptide further comprises a polypeptide linker between the        VH and VL domains that enables the sFv to form the desired        structure for antigen binding. For a review of sFv see Pluckthun        in The Pharmacology of Monoclonal Antibodies 113: 269-315        Rosenburg and Moore eds. Springer-Verlag, N.Y., 1994.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (VH) connected to a light chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161, and Hollinger et al., Proc. Natl.Acad Sci. USA 90: 6444-6448 (1993).

The invention also contemplates multivalent antibodies having at leasttwo binding domains. The binding domains may have specificity for thesame ligand or for different ligands. In one embodiment themultispecific molecule is a bispecific antibody (BsAb), which carries atleast two different binding domains, at least one of which is ofantibody origin. Multivalent antibodies may be produced by a number ofmethods. Various methods for preparing bi- or multivalent antibodies arefor example described in U.S. Pat. Nos. 5,260,203; 5,455,030; 4,881,175;5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.

The invention contemplate both polyclonal and monoclonal antibody,antigen binding fragments and recombinant proteins thereof which arecapable of binding an epitope according to the invention.

The preparation of polyclonal antibodies is well-known to those skilledin the art. See, for example, Green et al. 1992. Production ofPolyclonal Antisera, in: Immunochemical Protocols (Manson, ed.), pages1-5 (Humana Press); Coligan, et al., Production of Polyclonal Antiserain Rabbits, Rats Mice and Hamsters, in: Current Protocols in Immunology,section 2.4.1, which are hereby incorporated by reference.

The preparation of monoclonal antibodies likewise is conventional. See,for example, Kohler & Milstein, Nature, 256:495-7 (1975); Coligan, etal., sections 2.5.1-2.6.7; and Harlow, et al., in: Antibodies: ALaboratory Manual, page 726 ,Cold Spring Harbor Pub. (1988), Monoclonalantibodies can be isolated and purified from hybridoma cultures by avariety of well-established techniques. Such isolation techniquesinclude affinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, e.g., Coligan, etal., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes, et al.,Purification of Immunoglobulin G (IgG). In: Methods in MolecularBiology, 1992, 10:79-104, Humana Press, N.Y.

Methods of in vitro and in vivo manipulation of monoclonal antibodiesare well known to those skilled in the art. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler and Milstein,1975, Nature 256, 495-7, or may be made by recombinant methods, e.g., asdescribed in U.S. Pat. No. 4,816,567. The monoclonal antibodies for usewith the present invention may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., 1991,Nature 352: 624-628, as well as in Marks et al., 1991, J Mol Biol 222:581-597. Another method involves humanizing a monoclonal antibody byrecombinant means to generate antibodies containing human specific andrecognizable sequences. See, for review, Holmes, et al., 1997, J Immunol158:2192-2201 and Vaswani, et al., 1998, Annals Allergy, Asthma &Immunol 81:105-115.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional polyclonal antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In additional to their specificity, the monoclonal antibodiesare advantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (US 4,816,567); Morrison etal., 1984, Proc Natl Acad Sci 81: 6851-6855.

Methods of making antibody fragments are also known in the art (see forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, NY, 1988, incorporated herein by reference). Antibodyfragments of the present invention can be prepared by proteolytichydrolysis of the antibody or by expression in E. coli of DNA encodingthe fragment. Antibody fragments can be obtained by pepsin or papaindigestion of whole antibodies conventional methods. For example,antibody fragments can be produced by enzymatic cleavage of antibodieswith pepsin to provide a 5S fragment denoted F(ab′)₂. This fragment canbe further cleaved using a thiol reducing agent, and optionally ablocking group for the sulfhydryl groups resulting from cleavage ofdisulfide linkages, to produce 3.5S Fab′ monovalent fragments.Alternatively, an enzymatic cleavage using pepsin produces twomonovalent Fab′ fragments and an Fc fragment directly. These methods aredescribed, for example, in U.S. Pat. No. 4,036,945 and U.S. Pat. No.4,331,647, and references contained therein. These patents are herebyincorporated in their entireties by reference.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody. For example, Fv fragments comprise anassociation of V_(H) and V_(L) chains. This association may benoncovalent or the variable chains can be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde.Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (sFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains connected by an oligonucleotide.The structural gene is inserted into an expression vector, which issubsequently introduced into a host cell such as E. coli. Therecombinant host cells synthesize a single polypeptide chain with alinker peptide bridging the two V domains. Methods for producing sFvsare described, for example, by Whitlow, et al., 1991, In: Methods: ACompanion to Methods in Enzymology, 2:97; Bird et al., 1988, Science242:423-426; U.S. Pat. No. 4,946,778; and Pack, et al., 1993,BioTechnology 11:1271-77.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) are often involved in antigen recognition andbinding. CDR peptides can be obtained by cloning or constructing genesencoding the CDR of an antibody of interest. Such genes are prepared,for example, by using the polymerase chain reaction to synthesize thevariable region from RNA of antibody-producing cells. See, for example,Larrick, et al., Methods: a Companion to Methods in Enzymology, Vol. 2,page 106 (1991).

The invention contemplates human and humanized forms of non-human (e.g.murine) antibodies. Such humanized antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)that contain a minimal sequence derived from non-human immunoglobulin,such as the eitope recognising sequence. For the most part, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a nonhuman species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. Humanized antibody(es) containing a minimalsequence(s) of antibody(es) of the invention, such as a sequence(s)recognising an epitope(s) described herein, is one of the preferredembodiments of the invention.

In some instances, Fv framework residues of the human immunoglobulin arereplaced by corresponding non-human residues. Furthermore, humanizedantibodies may comprise residues that are found neither in the recipientantibody nor in the imported CDR or framework sequences. Thesemodifications are made to further refine and optimize antibodyperformance. In general, humanized antibodies will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see: Jones et al., 1986, Nature321, 522-525; Reichmann et al., 1988, Nature 332, 323-329; Presta, 1992,Curr Op Struct Biol 2:593-596; Holmes et al., 1997, J Immunol158:2192-2201 and Vaswani, et al., 1998, Annals Allergy, Asthma &Immunol 81:105-115.

The generation of antibodies may be achieved by any standard methods inthe art for producing polyclonal and monoclonal antibodies using naturalor recombinant fragments of a sequence selected from any of thesequences identified as SEQ ID NOs: 1-37, as an antigen. Such antibodiesmay be also generated using variants or fragments of SEQ ID NOs: 1-37.

The antibodies may also be produced in vivo by the individual to betreated, for example, by administering an immunogenic fragment accordingto the invention to said individual. Accordingly, the present inventionfurther relates to a vaccine comprising an immunogenic fragmentdescribed above.

The application also relates to a method for producing an antibody ofthe invention said method comprising a step of providing of animmunogenic fragment described above.

The invention relates both to an antibody, which is capable ofmodulating, such as enhancing or attenuating, biological function ofIL-4 in particular a function related to inflammation, and to anantibody, which can recognise and specifically bind to IL-4 withoutmodulating biological activity thereof.

The invention relates to use of the above antibodies for therapeuticapplications involving the modulation of activity of IL-4.

In one aspect the invention relates to the use of a pharmaceuticalcomposition comprising an antibody described above.

EXAMPLES Example 1

Four peptides derived from IL-4 were designed and synthesized (SEQ IDNOs:1-4). Mapping of the location of the peptides was performedemploying PyMOL™ software, based on PyMOL v0.99 (DeLano Scientific LLC,South San Francisco, Calif., U.S.A). This was done based on the crystalstructure of the ternary complex of human II4-II4r-II13ra, PDB ID: 3BPNand 3BPL (LaPorte et al., 2008).

IL-4 interacts with two fibronectin type III modules (FN3-1 and FN3-2)of the extracellular part of the IL-4Rα) (FIGS. 1 and 2). IL-4 interactswith two fibronectin type III modules (FN3-1 and FN3-2) of theextracellular part of IL-4Rα and γc (FIGS. 3 and 4).

Example 2

4 peptides derived from IL-4 were tested in a neurite outgrowth assaywhether they had any biological activity.

Cerebellar granular neurons (CGN) were prepared from 3 or 7 postnatal(P) day Wistar rats (Charles River, Sulzfeld, Germany or Taconic, Ejby,Denmark). Cerebella were cleared of meninges and blood vessels, roughlyhomogenized by chopping, and trypsinized with trypsin from Sigma-Aldrich(Brøndby, Denmark). The neurons were washed in the presence of DNAse 1and soybean trypsin inhibitor (Sigma-Aldrich), and cellular debris waspelleted by centrifugation before plating. For single-cell cultureexperiments, P7 CGNs were plated at a density of 10,000 cells/well ontouncoated eight-well Lab-Tek chamber slides (NUNC, Slangerup, Denmark) inNeurobasal-A medium supplemented with 0.4% (w/v) BSA. Peptides atvarious concentrations were added to the medium immediately afterplating, and cells were maintained at 37° C. and 5% CO₂ for 24 h.Cultures then were fixed, blocked and incubated with polyclonal rabbitantibody against rat GAP-43 (Chemicon, Temecula, Calif., USA) followedby incubation with secondary Alexa Fluor488 goat anti-rabbit antibody(Molecular Probes, Eugene, Oreg., USA) as previously described(Neiiendam et al., 2004). The immunostained cultures were all recordedby computer-assisted fluorescence microscopy using a Nikon Diaphotinverted microscope (Nikon, Japan) equipped with a Nikon Plane 20×objective. Images were captured with a charge-coupled device videocamera (Grundig Electronics, Nurnberg, Germany) using the softwarepackage Prima developed at the Protein Laboratory (University ofCopenhagen, Copenhagen, Denmark). The length of neuronal processes percell was estimated using the software package Process Length developedat the Protein Laboratory (Ronn et al. 2000). For estimation of neuriteoutgrowth, at least 200±20 cells were processed for each group in eachindividual experiment.

Results:

Peptides with the SEQ ID NOs: 1, 2, 3, and 4, from the IL-4 binding sitewere found to induce a neuritogenic response from primary neurons. Theresults of the effect of SEQ ID NO:1 2, 3 and 4 on cerebellar neuriteoutgrowth are shown in FIGS. 5, 6, 9, and 12, respectively.

Example 3

Primary macrophage cells (or cells of the AMJ2C8 macrophage cell line,see Ryan et al., 1997) can be cultured for 24 h at a density of 6×10⁻⁵cells/ml in 12-well plates (Nunc, Slangerup, Denmark) at 37° C., in 5%CO₂ and 95% humidity. For determination of TNF-α release in response toLPS stimulation, triplicate cultures were cultured in DMEM with 10% FCSfor 24 h and then stimulated with 0-10 μg/ml LPS for an additional 24 hperiod, after which culture supernatants were collected. Determinationof TNF-α concentrations in conditioned media from LPS-treatedmacrophages was done employing the L929 fibroblast-like cells which weresensitive to TNF-α upon exposure to actinomycin D (He et al., 2002).L929 cells were seeded in 96-well plates at a density of 20.000 cellsper well and maintained at 37° C., 5% CO₂, RPMI 1640 supplemented with10% FCS and 0.5% penicillin-streptomycin. At 1 h prior to use as theTNF-α bioassay, L929 cells were pre-treated with 5 μg/ml actrinomycin D(Sigma), and further incubated with conditioned medium, in variousdilutions, from LPS-treated macrophage cultures. Cell viability was thanevaluated using the CellTiter 96 assay (Promega, Madison, Wis., USA).

Macrophage Activation Test-System

-   -   Macrophages were seeded in 6 well multidish with 9.6 cm² per        well, in the density 10.000 cells/well.    -   Peptides or protein with potential anti-inflammatory effects        were added to the culture. As negative control, medium was added        to one well and as a positive control, 100 μM hydrocortisone was        added to one well.    -   Cell cultures were incubated for 24 h at 37° C.    -   IFN-γ was added to the macrophage cultures to activate        macrophages in the concentration 0.01 μg/ml. As control no IFN-γ        but medium was added to one well.    -   Fibroblast cells were seeded in a 96 well plate, in the        concentration 0.2×10² cells/ml.    -   Both cell cultures were incubated for 24 h at 37° C.

Conditioned medium from macrophages was collected by spinning the cellsolution for 5 min at 1200 rpm. The conditioned medium was added tofibroblasts, TNF-α was added for the titration curve and finallyactinomycin D was added to the fibroblasts in the concentration 0.5μg/ml.

Results:

The effect of peptides with SEQ ID NOs:1, 3, 5, 6, and 19 on inhibitionof an inflammatory response in macrophage cell cultures was tested.Results are shown in FIGS. 7, 10, and 13-17.

Example 4 Binding Studies Using Surface Plasmon Resonance (SPR) Analysis

Recombinant IL4Rα was immobilized on a CM5 sensor chip. Theimmobilization process was done by activating the carboxymethylateddextran matrix with 35 μl activation solution followed by an injectionof protein in 10 mM sodium acetate solution (pH 5.0). After a desiredlevel of protein was immobilized 35 μl of deactivation solution isinjected to deactivate any free carboxymethylated groups in the dextranmatrix. One flow cell was always empty as a control. Each analyte(recombinant IL-4 or IL-4-derived peptides) was diluted in PBS andinjected at a flow rate of 10 μl/min. The obtained data was analyzed byperforming a non-linear curve fitting using the software BIAevaluationv.4 from Biacore. The curves were fitted to a 1:1 Langmuir binding modelwhich describes the interaction of two molecules in 1:1 complex. Theaffinity constant (K_(D)) was calculated from the association rateconstant (k_(a)) and the dissociation rate constant (k_(d)). This wasdone by using the following formula, where L is the immobilized ligand,A the analyte, and LA is the analyte-ligand complex:

${{Langmuir}\mspace{14mu} 1\text{:}1\mspace{14mu} {model}\text{:}\mspace{11mu} L} + {\frac{\left. k_{a}\rightarrow \right.}{\left. \leftarrow k_{d} \right.}L\; A}$

Rate of Decreasing Ligand Concentration

$\frac{\lbrack L\rbrack}{t} = {- \left( {{k_{a}*\lbrack L\rbrack*\lbrack A\rbrack} - {k_{d}*\left\lbrack {L\; A} \right\rbrack}} \right)}$

Rate of Increasing Product Concentration

$\frac{\left\lbrack {L\; A} \right\rbrack}{t} = {{k_{a}*\lbrack L\rbrack*\lbrack A\rbrack} - {k_{d}*\left\lbrack {L\; A} \right\rbrack}}$

At Steady State

$\frac{\left\lbrack {L\; A} \right\rbrack}{t} = {\left. 0\Rightarrow{{k_{a}*\lbrack L\rbrack*\lbrack A\rbrack} - {k_{d}*\left\lbrack {L\; A} \right\rbrack}} \right. = {\left. 0\Rightarrow\frac{\lbrack L\rbrack*\lbrack A\rbrack}{\left\lbrack {L\; A} \right\rbrack} \right. = {\frac{k_{d}}{k_{a}} = K_{D}}}}$

Results:

Binding between Ph2 (SEQ ID NO:3), and IL4rα, and between Ph3 (SEQ IDNO:1) and IL4rα was studied. The results are shown in FIGS. 8 and 11,respectively.

REFERENCES

Agnello D, Lankford C S, Bream J, Morinobu A, Gadina M, O'Shea J J,Frucht D M. Cytokines and transcription factors that regulate T helpercell differentiation: new players and new insights. J Clin Immunol.2003, 23, 147-61.

Hage T, Sebald W, Reinemer P. Crystal structure of theinterleukin-4/receptor α chain complex reveals a mosaic bindinginterface. Cell 1999, 97, 271-281.

He B P, Wen W, Strong M J. Activated microglia (BV-2) facilitatation ofTNF-α-mediated motor meuron death in vitro. J Immunol. 2002, 128, 31-38.

Izuhara K, Arima K, Yasunaga S. IL-4 and IL13: Their patological rolesin allergic diseases and their potencial in developing new therapies.Curr Drug Targets-Inflam Allergy 2002, 1 263-269.

Leach M W, Mary Ellen Rybak M E, Rosenblum I Y. Safety Evaluation ofRecombinant Human Interleukin-4.Clin Immunol Immunopathol. 1997, 83,12-14.

Martin R. Interleukin 4 treatment of psoriasis: are pleiotropiccytokines suitable therapies for autoimmune diseases? TRENDS PharmacolSci. 2003, 24, 613-616.

Martinez F O, Sica A, Mantovani A, Locati M. Macrophage activation andpolarization. Front Biosci. 2008, 13, 453-61.

Muller T, Oehlenschlager F, Buehner M. Human interleukin-4 and variantR88Q: phasing X-ray diffraction data by molecular replacement usingX-ray and nuclear magnetic resonance models. 1995, 247, 360-372.

Neiiendam J, Køhler L, Christensen C, Li S, Pedersen M V, Ditlevsen D,Kornum M, Kiselyov V, Berezin V, Bock E. An NCAM-derived FGF-receptoragonist, the FGL-peptide, induces neurite outgrowth and neuronalsurvival in primary rat neurons. J. Neurochem. 2004, 91, 920-935.

LaPorte S L, Juo Z S, Vaclavikova J, CoIf L A, Qi X, Heller N M, KeeganA D, Garcia K C. Molecular and structural basis of cytokine receptorpleiotropy in the interleukin-4/13 system. Cell 2008, 132, 259-272.

Rønn L C B, Ralets I, Hartz B, Bech M, Berezin A, Berezin V, Moller A,Bock, E A simple procedure for quantification of neurite outgrowth basedon stereological principles. J. Neurosci. Methods 2000, 100, 25-32.

-   Ryan L K, Colenbock D T, Wu J, Vermeulen M W. Characterization of    proinflammatory cytokine production and CD14 expression by murine    alveolar macrophage cell lines. In Vitro Cell Dev Biol Anim. 1997,    33, 647-653.

Soroka V, Kiryushko D, Novitskaya V, Ronn L C, Poulsen F M, Holm A, BockE and Berezin V. Induction of neuronal differentiation by a peptidecorresponding to the homophilic binding site of the second Ig module ofNCAM. J. Biol. Chem. 2002, 277, 24676-24683.

Szegedi A, Aleksza M, Gonda A, Irinyi B, Sipka S, Hunyadi J,Antal-Szalmás P. Elevated rate of Thelperl (T(H)1) lymphocytes and serumIFN-gamma levels in psoriatic patients. Immunol Lett. 2003, 86, 277-80.

Whitehead R P, Unger J M, Goodwin J W, Walker M J, Thompson J A,Flaherty L E, Sondak V K. Phase II trial of recombinant humaninterleukin-4 in patients with disseminated malignant melanoma: aSouthwest Oncology Group study. J Imminother. 1998, 21, 440-446.

1. A compound comprising a peptide consisting of at most 35 contiguousamino acid residues derived from interleukin-4 (SEQ ID NO:38), or afragment thereof or a variant being at least 70% identical to SEQ IDNO:38 or a fragment thereof.
 2. A compound according to claim 1, whereinsaid peptide comprises a sequence with the formula X1—X2—X3, wherein X1is L, X2 is I, Q, G, T, or a charged amino acid; and X3 is Q, T, or acharged amino acid.
 3. A compound according to claim 2, wherein X2 is Ior Q.
 4. A compound according to claim 3, wherein X2 is I.
 5. A compoundaccording to claim 3, wherein X2 is Q.
 6. A compound according to claim2, wherein X2 is a charged amino acid.
 7. A compound according to any ofclaims 2-6, wherein X3 is a charged amino acid.
 8. A compound accordingto claim 7, wherein X3 is R or E.
 9. A compound according to claim 8,wherein X3 is R.
 10. A compound according to claim 8, wherein X3 is E.11. A compound according to any of claims 2-6, wherein X3 is Q or T. 12.A compound according to claim 2, wherein X1 is L, X2 is I, and X3 is R.13. A compound according to claim 2, wherein X1 is L, X2 is Q and X3 isE.
 14. A compound according to any of the preceding claims, wherein saidpeptide is capable of interacting with the IL-4 receptor.
 15. A compoundaccording to any of the preceding claims, wherein said peptide comprisesa part of an alpha-helix of IL-4.
 16. The compound according to any ofthe preceding claims, wherein said peptide is capable of modulating IL-4receptor signalling.
 17. The compound according to claim 16, whereinsaid peptide is capable of stimulating IL-4 receptor signalling.
 18. Thecompound according to claim 16, wherein said peptide is capable ofinhibiting IL-4 receptor signalling.
 19. The compound according to anyof the preceding claims, wherein said peptide comprises one of thefollowing sequences AQFHRHKQLIRFLKRA (SEQ ID NO: 1) AITLQEIIKTLNSA(SEQ ID NO: 2) ARFLKRLDRNLWGG (SEQ ID NO: 3) AERLKTIMREKYSKS(SEQ ID NO: 4) LQEIKTLN (SEQ ID NO: 5) KRLQQNLFGG (SEQ ID NO: 6)Ac-AQFHRHKQLIRFLKRA (SEQ ID NO: 7) QEIIKKL (SEQ ID NO: 8) AIQNQEEIKYLNS(SEQ ID NO: 9) AIILQEI (SEQ ID NO: 10) IVLQEII (SEQ ID NO: 11)TLGEIIKGVNS (SEQ ID NO: 12) VTLIDHSEEIFKTLN (SEQ ID NO: 13) LQERIKSLN(SEQ ID NO: 14) RLDRENVAVYNLW (SEQ ID NO: 15) LRSLDRNL (SEQ ID NO: 16)RLLRLDRN (SEQ ID NO: 17) RFLKRYFYNLEENL (SEQ ID NO: 18) RNKQVIDSLAKFLKR(SEQ ID NO: 19) RHKALIR (SEQ ID NO: 20) KKLIRYLK (SEQ ID NO: 21) RHKTLIR(SEQ ID NO: 22) MQDKYSKS (SEQ ID NO: 23) AERVKIEQREYKKYS (SEQ ID NO: 24)SQLIRFLKRLA (SEQ ID NO: 25) TVTDIFAASKNTT (SEQ ID NO: 26) TLENFLERLKTA(SEQ ID NO: 27) TEKEVLRQFYSA (SEQ ID NO: 28) KTLTELTKTLNS(SEQ ID NO: 29) AHKEIIKTLNSLQKA (SEQ ID NO: 30) AKTLSTELTVTA(SEQ ID NO: 31) STLENFLERLA (SEQ ID NO: 32) NEERLKTIMRA (SEQ ID NO: 33)RAATVLRQFYSR (SEQ ID NO: 34) KTLNSLTEQKT (SEQ ID NO: 35) AHRHKQLIRA(SEQ ID NO: 36) ATAQQFHRHKQA, (SEQ ID NO: 37)

or a fragment or a variant thereof.
 20. The compound according to claim12, wherein said peptide comprises one of the following sequencesAQFHRHKQLIRFLKRA (SEQ ID NO: 1) Ac-AQFHRHKQLIRFLKRA (SEQ ID NO: 7)RHKALIR (SEQ ID NO: 20) KKLIRYLK (SEQ ID NO: 21) RHKTLIR (SEQ ID NO: 22)SQLIRFLKRLA (SEQ ID NO: 25) AHRHKQLIRA, (SEQ ID NO: 36)

or a fragment or a variant thereof.
 21. The compound according to claim13, wherein said peptide comprises one of the following sequencesAITLQEIIKTLNSA (SEQ ID NO: 2) LQEIKTLN (SEQ ID NO: 5) AIILQEI(SEQ ID NO: 10) IVLQEII (SEQ ID NO: 11) LQERIKSLN (SEQ ID NO: 14)AHKEIIKTLNSLQKA, (SEQ ID NO: 30)

or a fragment or a variant thereof.
 22. The compound according to any ofthe preceding claims, wherein said peptide is capable of activatingB-cells.
 23. The compound according to any of the preceding claims,wherein said peptide is capable of activating growth and survival ofT-cells.
 24. The compound according to any of the preceding claims,wherein said peptide is capable of downregulating C5a and C3a inmonocytes and dendritic cells.
 25. The compound according to any of thepreceding claims, wherein said peptide is capable of inhibitingmacrophage activation.
 26. An isolated peptide sequence consisting ofone of the following sequences AQFHRHKQLIRFLKRA (SEQ ID NO: 1)AITLQEIIKTLNSA (SEQ ID NO: 2) ARFLKRLDRNLWGG (SEQ ID NO: 3)AERLKTIMREKYSKS (SEQ ID NO: 4) LQEIKTLN (SEQ ID NO: 5) KRLQQNLFGG(SEQ ID NO: 6) Ac-AQFHRHKQLIRFLKRA (SEQ ID NO: 7) QEIIKKL (SEQ ID NO: 8)AIQNQEEIKYLNS (SEQ ID NO: 9) AIILQEI (SEQ ID NO: 10) IVLQEII(SEQ ID NO: 11) TLGEIIKGVNS (SEQ ID NO: 12) VTLIDHSEEIFKTLN(SEQ ID NO: 13) LQERIKSLN (SEQ ID NO: 14) RLDRENVAVYNLW (SEQ ID NO: 15)LRSLDRNL (SEQ ID NO: 16) RLLRLDRN (SEQ ID NO: 17) RFLKRYFYNLEENL(SEQ ID NO: 18) RNKQVIDSLAKFLKR (SEQ ID NO: 19) RHKALIR (SEQ ID NO: 20)KKLIRYLK (SEQ ID NO: 21) RHKTLIR (SEQ ID NO: 22) MQDKYSKS(SEQ ID NO: 23) AERVKIEQREYKKYS, (SEQ ID NO: 24)

or a fragment or a variant thereof.
 27. A pharmaceutical compositioncomprising at least one compound as defined in claims 1-25.
 28. Use of acompound according to any of claims 1-25 or a pharmaceutical compositionaccording to claim 27 for the manufacturing of a medicament.
 29. The useaccording to claim 28, wherein the medicament is for treatment ofdiseases or conditions wherein modulation of IL-4 receptor signalling isessential.
 30. The use according to claim 28, wherein the medicament isfor treatment of inflammatory diseases or conditions.
 31. The useaccording to claim 30, wherein the medicament is for treatment of anautoimmune disease or condition.
 32. The use according to claim 30,wherein the medicament is for treatment of rheumatoid arthritis.
 33. Theuse according to any of claims 28-32, wherein the medicament is forsubcutaneous, intravenous, oral, nasal, pulmonal, topical administrationor parental administration supplemented with intraarticularadministration into or near joint capsules.
 34. Use of a compoundaccording to any of claims 1-25 for the production of an antibody. 35.An antibody capable of binding an epitope comprising at least one of theamino acid sequences SEQ ID NO:s 1-37.
 36. An antibody according toclaim 35, wherein said antibody is capable of modulating biologicalactivity mediated by IL-4.
 37. Use of an antibody according to claims35-36 for the manufacture of a medicament for treatment of aninflammatory disease or condition.
 38. A pharmaceutical compositioncomprising an antibody according to any of claims 35-36.
 39. Method oftreatment comprising administering to an individual in need an effectiveamount of a compound according to any of the claims 1-25, an antibodyaccording to claim 35, or pharmaceutical composition according to claim27 or 38.